Galactose is an abundant sugar in many different raw materials for industrial fermentation processes. Thus, galactose is one of the sugars present in lactose, the primary sugar of whey. It is also present in raffinose, a sugar present in beet molasses. Furthermore, it represents 3-18% of the sugars present in hemicellulose from various plant sources. Galactose can be metabolized by most micro-organisms. However, the rate of uptake of galactose is for most organisms substantially lower than the uptake of the sugars glucose, fructose and mannose. For exploitation of galactose as a raw material for industrial fermentations in the production of low value added products like ethanol, lactic acid and citric acid the slow uptake rate of galactose represents a fundamental problem.
Within the last decade metabolic engineering has been successfully applied for optimisation of several industrial fermentation processes (Nielsen, 2001). Recombinant DNA techniques have facilitated the ability to genetically modify suitable host systems, and this has resulted in recombinant strains that have reduced by-product formation with a resulting increase in the overall yield of product. For many industrial processes—especially those involving low-value added products—the yield is certainly important, but it is also important to have a high rate of conversion of the substrate into the product. This requires optimisation of the flux through the central carbon metabolism, which has been attempted for glycolysis in Saccharomyces cerevisiae (Schaff et al., 1989) and in other micro-organisms. These attempts have, however, largely failed for two major reasons. First, control of flux through central carbon metabolism is often distributed over many enzymes, so an increase in flux requires increased activity of many (or all) enzymes in the pathway. Second, regulation of glycolysis—both at the genetic and at the enzymatic level—is generally believed to be very rigid making it difficult to modulate flux through amplification of individual enzyme activities.
One way to solve these problems may be over expression of many (or all) genes encoding the enzymes in a given pathway. However, this may impose a physiological burden on the cell by draining pools of nucleotides or amino acids, or by slowing down transcriptional or translational efficiency. This may have metabolic consequences in other parts of metabolism, which negatively affects the overall performance of the cell. Furthermore, high levels of all the enzymes in the pathway may lead to significant changes in metabolite levels, which may result in down-regulation of some enzymes.
In fungi the uptake and metabolism of galactose is via the Leloir pathway (see FIG. 1). In this pathway galactose is first transported into the cell by a specific permease. In the next step the sugar is phosphorylated into galactose-1-phosphate, a reaction that is catalyzed by a specific kinase. In the next step of the pathway galactose-1-phosphate reacts with UDP-glucose and forms glucose-1-phosphate and UDP-galactose, a reaction that is catalyzed by galactose-1-phosphate uridylyltransferase. Regeneration of UDP-glucose from UDP-galactose is catalyzed by a separate enzyme involved in the pathway—UDP-glucose 4-epimerase. Glucose-1-phosphate is the end product of the Leloir pathway, but in the further catabolism of galactose this sugar phosphate is further converted into glucose-6-phosphate, which may be further processed via the Embden Meyerhof Parnas pathway or the Pentose Phosphate pathway. The conversion of glucose-1 phosphate to glucose-6 phosphate is catalyzed by the enzyme phosphoglucomutase (PGM). As glucose-1 phosphate is used as precursor for trehalose, glycogen and glucan biosynthesis, PGM also plays a role during metabolism of glucose, but here it catalyzes the conversion of glucose-6 phosphate to glucose-1 phosphate.
The galactose metabolism is subjected to dual control, being induced by galactose and repressed by glucose. Regulation has been extensively studied in the yeast Saccharomyces cerevisae, which preferably uses glucose as energy and carbon source over galactose. In this yeast the genes involved in galactose metabolism, often referred to as the GAL genes, are subjected to glucose repression to a much larger extent than other genes controlled by glucose such as the MAL genes and the SUC genes (Johnston and Carlson, 1992; Klein et al., 1998). The structural GAL genes subjected to this dual control are the GAL2 gene, encoding galactose permease, the GAL1 gene, encoding galactokinase, the GAL7 gene, encoding galactose-1-phosphate uridylyltransferase, and the GAL10 gene, encoding UDP-glucose 4-epimerase. Transcription of these structural GAL genes is enhanced 1000-fold after de-repression by glucose and induction by galactose (Melcher, 1997). In S. cerevisiae phosphoglucomutase is often referred to as the Gal5 protein, but as there are two isoforms of the enzyme a more correct description of this enzyme is designation of each isoform encoded by the two genes PGM1 and PGM2. Both these genes are not under the same tight control as GAL1, GAL2, GAL7 and GAL10, as their transcription are only increased threefold in the presence of galactose (Oh and Hopper, 1990). This is likely a consequence of the high basic level of expression of the PGM2 gene at non-induced conditions independent of the positive transcriptional activator Gal4 (Oh and Hopper, 1990), which may be due to the role of phosphoglucomutase in the glycogen, trehalose and glucan biosynthesis. In fact phosphoglucomutase is generally believed to be in excess as it can equilibrate the pools of glucose-1 phosphate and glucose-6 phosphate (Zubay, 1988). Furthermore this enzyme has a high affinity for its substrates (Daugherty et al. 1975). For these reasons gene products of PGM1 and PGM2 are generally not believed to exert any degree of flux control in the Leloir pathway, which is consistent with the situation of the glycolysis where no single enzyme has been shown to exert any significant flux control.
The system involved in regulation of the expression of the structural GAL genes is illustrated in FIG. 2. The regulatory GAL gene products comprise Gal3 and Gal4 that are necessary for induction of the GAL genes. The protein Gal4 acts as a transcriptional activator of the GAL genes by binding to specific sequences upstream of the coding region (Johnston, 1987). The GAL80 gene encodes a protein that binds to Gal4 and prevents this protein from activating transcription. Recent in vitro studies strongly indicated that Gal3 interacts with Gal80 and the interaction of Gal3 with Gal80 is believed to relieve the interaction between Gal80 and Gal4 which covers the activating domain of Gal4, and hence, transcriptional activation of the GAL genes is possible (Platt and Reece, 1998). Another regulatory gene recently devoted to the group of GAL genes is the GAL6 gene, which has been shown to be regulated by galactose (Zheng et al., 1997). GAL6 is regulated in similar way as GAL80 and the gene product has a negative impact on expression of the GAL genes, but the actual mechanism of action within the GAL system still remains to be elucidated. Finally, Mig1-mediated glucose repression also imposes control over the GAL system (Johnston and Carlson, 1992). The Mig1 protein takes part in a protein complex with the proteins Ssn6 and Tup1, which binds to the GAL1 promoter and the GAL4 promoter (Keleher et al., 1992; Treitel and Carlson, 1995; Frovola et al., 1999) and hence preventing transcription of these two genes. Repression of the latter gene has a major effect by down-regulation of the whole GAL system.
Ostergaard et al. (2000) report on modulation of the regulatory GAL system for improving the uptake and metabolism of galactose. They constructed and analysed a number of recombinant strains with alteration in the expression of regulatory genes. The best effect was obtained by deleting the three genes MIG1, GAL80 and GAL6 whereby the galactose uptake rate increases 41%. A positive effect was also found by over expression of GAL4 resulting in a 26% increase in the galactose uptake rate. Deletion of GAL6 alone results in a 24% increase in the galactose uptake rate, whereas deletion of MIG1 and GAL80 alone increases the galactose utilisation with 15% (Ostergaard et al., 2000). Based on these findings it was speculated that deletion of negative regulatory genes like MIG1, GAL80 and GAL6 and over expression of the gene GAL4 encoding the positive transcriptional regulator results in an co-ordinated and balanced increased expression of the structural GAL genes GAL1, GAL2, GAL 7 and GAL10 leading to an increased flux through the Leloir pathway.
Masuda et al (2001) investigated the effect of lithium on the metabolism of galactose in Saccharomyces cerevisiae and in particular on the phosphoglucomutase activity due to transcription of the PGM2 gene. One of the constructs they used was an auxotrophic (leucine and histidine requiring) strain over expressing PGM2. Such a strain would be unsuitable for commercial use because of its auxotrophic nature and would not be able to grow on minimal media (i.e. media not supplemented to supply the amino acids required by the auxotrophic strain. This strain is used in Masuda et al. only as a basis for comparison in the investigation of the effect of lithium with a view to better understanding the mechanism of the therapeutic effect of lithium in treating manic depressive disorder in humans. PGM2 over expression was used only to demonstrate that this could overcome the suppression of PGM2 activity otherwise produced by lithium stress. No beneficial result relevant to the normal cultivation of S. cerevisiae was reported.
In the present invention we applied genome-wide transcription analysis using DNA arrays to identify differently expressed genes in three of the strains with different galactose utilisation capacity studied by Ostergaard et al. (2000). Based on this analysis we identified PGM2 to be an important gene for ensuring a high galactose uptake rate.